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Technical Abstract: Plum pox potyvirus (PPV) has been recognized in the US since 1999. The disease outbreak is limited to a four county area in southern Pennsylvania. Using sequencing and phylogenetic analyses, we have determined that the Pennsylvania isolates divide into two clades, suggesting the possibility of multiple introductions. The two clades have somewhat distinct biological properties, further supporting the phylogenetic divisions. To provide context for the overall Pennsylvania population diversity, several isolates from a single orchard were sequenced. In contrast to the populations taken from around the quarantine zone, the isolates from single orchards were remarkably identical. PPV diversity levels also were determined for replicating populations within individual hosts. Like most RNA viruses, PPV generates high levels of genetic diversity within its replicating populations, with mutation frequencies ranging from 0.8 to 1.3 mutations/1000 bases. However, very little of this diversity is observed in the consensus sequences of isolates found within close proximity in a single orchard, suggesting that most of the population diversity observed at the level of replicating populations has little effect in the natural evolutionary processes of PPV.